Shiga toxins 1 and 2 (Stx1/2) are the primary virulence factors of Shiga toxin-producing E. coli (STEC), which are major food-, and waterborne pathogens afflicting both developed and developing countries. An estimated 265,000 STEC infections occur annually in the US. In addition to severe and often bloody diarrhea, the life-threatening complication hemolytic uremic syndrome (HUS) occurs in 2-7% of victims (primarily children). No STEC vaccines or therapies beyond supportive care are available. Antibiotics are not used since they can increase toxin production and risk of HUS. The severity of STEC infection, propensity for outbreaks, and lack of effective treatments make STEC concerning potential bioterror agents and a public health priority. Here, we describe an innovative strategy to discover D-peptide inhibitors of Stx1/2 to combat STEC infection. D-peptides, the mirror images of natural L-peptides, cannot be digested by proteases and, therefore, have the potential for long in vivo half-lives and low immunogenicity. They can readily disrupt protein interfaces with high potency and specificity compared to small molecules and are much less expensive to produce than antibodies. D-peptides are ideal candidates for Stx1/2 neutralization in the gut and/or systemic circulation. Navigen's drug discovery platform employs an enantiomeric screening technology (mirror-image phage display) coupled with protein design. We have successfully validated this platform technology by identifying D- peptide inhibitors of HIV, RSV, and Ebola. Our anti-HIV D-peptide was the first potent and specific D-peptide inhibitor to be discovered and is in advanced preclinical trials. It binds a functionally critical and conserved hydrophobic pocket on HIV's trimeric surface glycoprotein, gp41. A trimeric version of this D-peptide binds to all three gp41 pockets, providing a strong avidity boost. Stx1/2 is each composed of a single enzymatically active A subunit and five receptor-binding B subunits that form a pentameric ring. Each of the B pentamer subunits contains a vulnerable pocket analogous to those of our viral targets. The Stx1/2 B subunit pockets are excellent targets for us to next apply our expertise in D- peptide drug design given 1) our success in targeting analogous pockets at functionally critical interfaces, 2) the pentameric Stx1/2 target, which inspires design of pentameric D-peptides with strong avidity, and 3) the likelihood of D-peptide stability and activity in the gut without disturbing native flora. Furthermore, the B subunits of Stx1 and Stx from Shigella dysenteriae are identical, enabling dual therapeutic use for an anti- Stx1B D-peptide. These benefits have generated strong enthusiasm for this project from GI infectious disease clinicians, who understand the dramatic potential impact an anti-Stx1/2 D-peptide would have in the clinic. In this two-year grant, we propose to discover, structurally characterize, and optimize pentameric D-peptide inhibitors that will neutralize Stx1/2 with high potency. Success in this project will launch a new class of inhibitors against pathogenic bacteria important to global health.